Identification of HSD17B12 as an enzyme catalyzing drug reduction reactions through investigation of nabumetone metabolism.

Nabumetone, a nonsteroidal anti-inflammatory prodrug, is converted to a pharmacologically active metabolite, 6-methoxy-2-naphthylacetic acid (6-MNA); however, it is 11-fold more efficiently converted to 4-(6-methoxy-2-naphthyl)butan-2-ol (MNBO) via a reduction reaction in human hepatocytes. The goal of this study was to identify the enzyme(s) responsible for MNBO formation from nabumetone in the human liver. MNBO formation by human liver microsomes (HLM) was 5.7-fold higher than in the liver cytosol. In a panel of 24 individual HLM samples with quantitative proteomics data, the 17ß-hydroxysteroid dehydrogenase 12 (HSD17B12) protein level had the high correlation coefficient (r = 0.80, P < 0.001) among 4457 proteins quantified in microsomal fractions during MNBO formation. Recombinant HSD17B12 expressed in HEK293T cells exhibited prominent nabumetone reductase activity, and the contribution of HSD17B12 to the activity in the HLM was calculated as almost 100%. MNBO formation in HepG2 and Huh7 cells was significantly decreased by the knockdown of HSD17B12. We also examined the role of HSD17B12 in drug metabolism and found that recombinant HSD17B12 catalyzed the reduction reactions of pentoxifylline and S-warfarin, suggesting that HSD17B12 prefers compounds containing a methyl ketone group on the alkyl chain. In conclusion, our study demonstrated that HSD17B12 is responsible for the formation of MNBO from nabumetone. Together with the evidence for pentoxifylline and S-warfarin reduction, this is the first study to report that HSD17B12, which is known to metabolize endogenous compounds, such as estrone and 3-ketoacyl-CoA, plays a role as a drug-metabolizing enzyme.

Nabumetone induced photogenotoxicity mechanism mediated by ROS generation under environmental UV radiation in human keratinocytes (HaCaT) cell line.

Nabumetone (NB) is a non-steroidal anti-inflammatory drug (NSAID), prescribed for managing pain associated with acute/chronic rheumatoid arthritis, osteoarthritis and other musculoskeletal disorders. Though some incidences of photosensitivity have been reported, there is limited information available on its phototoxicity potential. In this study, NB photodegraded in a time-dependant manner (0-4 h) under UVA (1.5 mW/cm2), UVB (0.6 mW/cm2) and natural sunlight as observed through UV-vis spectrophotometer and the results were further confirmed with Ultra High-Performance Liquid Chromatography (UHPLC). Photosensitized NB generated reactive oxygen species (ROS) as observed by lipid peroxidation, suggesting oxidative degradation of lipids in cell membrane, thereby resulting in cell damage. MTT and NRU (neutral red uptake) assays revealed that NB induced phototoxicity in concentration-dependent manner (0.5, 1, 5, 10 µg/ml) under UVA, UVB and sunlight exposure (30 min) in human keratinocytes cell line (HaCaT), with significant phototoxicity at the concentration of 5 µg/ml. Photosensitized NB generated intracellular ROS, disrupted mitochondrial and lysosomal membrane integrity, resulting in cell death. UV-induced genotoxicity by NB was confirmed through micronuclei generation, γ-H2AX induction and cyclobutane pyrimidine dimer formation. This is the first study which showed the phototoxicity and photogenotoxicity potential of NB in HaCaT cell line. We also observed that photosensitized NB upregulated inflammatory markers, such as COX-2 and TNFα. This study proposes that sunlight exposure should be avoided by patients using nabumetone and proper guidance should be provided by clinicians regarding photosensitivity of drugs for better safety and efficacy.

Role of human flavin-containing monooxygenase (FMO) 5 in the metabolism of nabumetone: Baeyer-Villiger oxidation in the activation of the intermediate metabolite, 3-hydroxy nabumetone, to the active metabolite, 6-methoxy-2-naphthylacetic acid in vitro.

Nabumetone (NAB) is a non-steroidal anti-inflammatory drug used clinically, and its biotransformation includes the major active metabolite 6-methoxy-2-naphthylacetic acid (6-MNA). One of the key intermediates between NAB and 6-MNA may be 3-hydroxy nabumetone (3-OH-NAB). The aim of the present study was to investigate the role of flavin-containing monooxygenase (FMO) isoform 5 in the formation of 6-MNA from 3-OH-NAB. To elucidate the biotransformation of 3-OH-NAB to 6-MNA, an authentic standard of 3-OH-NAB was synthesised and used as a substrate in an incubation with human liver samples or recombinant enzymes. The formation of 3-OH-NAB was observed after the incubation of NAB with various cytochrome P450 (CYP) isoforms. However, 6-MNA itself was rarely detected from NAB and 3-OH-NAB. Further experiments revealed a 6-MNA peak derived from 3-OH-NAB in human hepatocytes. 6-MNA was also detected in the extract obtained from 3-OH-NAB by a combined incubation of recombinant human FMO5 and human liver S9. We herein demonstrated that the reaction involves carbon-carbon cleavage catalyzed by the Baeyer-Villiger oxidation (BVO) of a carbonyl compound, the BVO substrate, such as a ketol, by FMO5. Further in vitro inhibition experiments showed that multiple non-CYP enzymes are involved in the formation of 6-MNA from 3-OH-NAB.

Toward Multitasking Pharmacological COX-Targeting Agents: Non-Steroidal Anti-Inflammatory Prodrugs with Antiproliferative Effects.

The antitumor activity of certain anti-inflammatory drugs is often attributed to an indirect effect based on the inhibition of COX enzymes. In the case of anti-inflammatory prodrugs, this property could be attributed to the parent molecules with mechanism other than COX inhibition, particularly through formulations capable of slowing down their metabolic conversion. In this work, a pilot docking study aimed at comparing the interaction of two prodrugs, nabumetone (NB) and its tricyclic analog 7-methoxy-2,3-dihydro-1H-cyclopenta[b]naphthalen-1-one (MC), and their common active metabolite 6-methoxy-2-naphthylacetic acid (MNA) with the COX binding site, was carried out. Cytotoxicity, cytofluorimetry, and protein expression assays on prodrugs were also performed to assess their potential as antiproliferative agents that could help hypothesize an effective use as anticancer therapeutics. Encouraging results suggest that the studied compounds could act not only as precursors of the anti-inflammatory metabolite, but also as direct antiproliferative agents.

The association between nonsteroidal anti-inflammatory drugs and skin cancer: Different responses in American and European populations.

OBJECTIVE: To conduct a comprehensive systematic meta-analysis investigating the association of nonsteroidal anti-inflammatory drugs (NSAIDs) and their subtypes with skin cancer (SC) and its subclasses (basal cell carcinoma BCC; squamous cell carcinoma SCC; melanoma; nonmelanoma skin cancer NMSC) in general, American and European populations. METHODS: PubMed, Embase, the Cochrane Library, the China National Knowledge Infrastructure and ClinicalTrials.gov were searched up to 24 February 2019. Pooled effect sizes and 95% confidence intervals were used to estimate associations. RESULTS: Results based on 26 original studies including 223,619 cases and 1,398,507 controls showed both NSAIDs and nonselective Cyclooxygenase (COX) inhibitors to be statistically significantly associated with a reduced risk of SC, BCC, SCC and NMSC but not with melanoma. Conversely, no association was observed between selective Cyclooxygenase 2 (COX-2) inhibitors and SC or its subclasses. Further subgroup analysis showed that the results analyzed for American populations were almost the same as those for the general population. For European populations, neither NSAIDs nor its subtypes correlated significantly with susceptibility to SC or its subclasses. CONCLUSIONS: The use of NSAIDs might reduce the risk of SC, but many factors including study population, drug subtype, and disease subclass affect the significance of the association.

A metabolic pathway for the prodrug nabumetone to the pharmacologically active metabolite, 6-methoxy-2-naphthylacetic acid (6-MNA) by non-cytochrome P450 enzymes.

The pathway for the transformation of the prodrug nabumetone, 4-(6-methoxynaphthalen-2-yl)butan-2-one, to the active metabolite 6-methoxy-2-naphthylacetic acid (6-MNA), a potent cyclooxygenase-2 inhibitor, has not yet been clarified in humans.To confirm the activation pathway, authentic standards of the nabumetone intermediates, 2-(6-methoxynaphthalen-2-yl)ethyl acetate (6-MNEA), 2-(6-methoxynaphthalen-2-yl)ethan-1-ol (6-MNE-ol) and 2-(6-methoxynaphthalen-2-yl)acetaldehyde (6-MN-CHO) were synthesized. High performance liquid-chromatography and gas chromatography-mass spectrometry on nabumetone oxidation revealed the generation of three metabolites.The formation of 6-MNA after a 60-min incubation of nabumetone was detected and 6-MNE-ol, an alcohol-related intermediate, was also generated by in cryopreserved hepatocytes. However, 6-MNA was below detection limit, but 4-(6-methoxynaphthalen-2-yl)butan-2-ol (MNBO) and 4-(6-hydroxynaphthalen-2-yl)butan-2-one (M3) peak were found in both the microsomes and S9 extracts with any cofactors.Nabumetone has recently been proposed as a typical substrate of flavin-containing monooxygenase isoform 5 (FMO5) and was shown to be efficiently oxidized in vitro to 6-MNEA. 6-MNA was detected in the extract obtained from a combined incubation of recombinant FMO5 and S9 fractions.The specificity of FMO5 towards catalyzing this Baeyer-Villiger oxidation (BVO) was demonstrated by the inhibition of the BVO substrate, 4-methoxyphenylacetone. Further in vitro inhibition studies demonstrated that multiple non-cytochrome P450 enzymes are involved in the formation of 6-MNA.

Gut microbiota metabolizes nabumetone in vitro: Consequences for its bioavailability in vivo in the rodents with altered gut microbiome.

1. The underlying microbial metabolic activity toward xenobiotics is among the least explored factors contributing to the inter-individual variability in drug response. 2. Here, we analyzed the effect of microbiota on a non-steroidal anti-inflammatory drug nabumetone. 3. First, we cultivated the drug with the selected gut commensal and probiotic bacteria under both aerobic and anaerobic conditions and analyzed its metabolites by high-performance liquid chromatography (HPLC) with UV detection. To analyze the effect of microbiota on nabumetone pharmacokinetics in vivo, we administered a single oral dose of nabumetone to rodents with intentionally altered gut microbiome - either rats treated for three days with the antibiotic imipenem or to germ-free mice. Plasma levels of its main active metabolite 6 methoxy-2-naphthylacetic acid (6-MNA) were analyzed at pre-specified time intervals using HPLC with UV/fluorescence detection. 4. We found that nabumetone is metabolized by bacteria to its non-active metabolites and that this effect is stronger under anaerobic conditions. Although in vivo, none of the pharmacokinetic parameters of 6-MNA was significantly altered, there was a clear trend towards an increase of the AUC, Cmax and t1/2 in rats with reduced microbiota and germ-free mice.

Determination of the Absolute Configuration of the Nabumetone Metabolite 4-(6-Methoxy-2-naphthyl)butan-2-ol Using the Chiral Derivatizing Agent, 1-Fluoroindan-1-carboxylic Acid.

The absolute configuration of (+)-4-(6-methoxy-2-naphthyl)butan-2-ol ((+)-MNBO), a nabumetone metabolite, was determined using 1-fluoroindan-1-carboxylic acid (FICA). Both enantiomers of the FICA methyl esters were derivatized to diastereomeric esters of (+)-MNBO by an ester exchange reaction. The results of 1H- and 19F-NMR spectroscopy of the diastereomeric FICA esters of (+)-MNBO confirmed the absolute configuration of (+)-MNBO was (S).

Crystal structure of phospholipase A2 in complex with 1-naphthaleneacetic acid.

Phospholipase A2 (PLA2 ) is one of the rate limiting enzymes involved in the production of arachidonic acid, a potent inflammatory mediator. PLA2 is widely distributed all over the animal kingdom. It is also seen in inflammatory exudation and venoms of different organisms. The studies demonstrated that PLA2 inhibitors have broad spectrum activities that they can either be used against inflammation or envenomation. In this study, the inhibitory activity of 1-napthaleneacetic acid (NAA) against porcine pancreatic PLA2 has been explained through isothermal titration calorimetry and enzyme kinetics studies. The atomic level of interactions of NAA with PLA2 was also studied using X-ray crystallography. Apart from these findings, the theoretical binding affinities and mode of interactions of two naphthalene-based NSAIDs such as naproxen (NAP) and nabumetone (NAB) were studied through molecular modeling. The studies proved that the selected ligands are binding at the doorway of the active site cleft and hindering the substrate entry to the active site. The study brings out a potential scaffold for the designing of broad spectrum PLA2 inhibitors which can be used for inflammation or envenomation. © 2018 IUBMB Life, 70(10):995-1001, 2018.

Pharmacokinetic and Pharmacodynamic Interaction of Andrographolide and Standardized Extract of Andrographis paniculata (Nees) with Nabumetone in Wistar Rats.

The aim of the study was to investigate the herb-drug interaction of Andrographis paniculata Nees (Acanthaceae) and Andrographolide (AN) with nabumetone (NAB) in wistar rats. Pharmacokinetic and pharmacodynamic interactions were studied after co-administration of APE and AN with NAB in Wistar rats. In pharmacokinetic studies, significant decrease in Cmax, AUC0-t and AUC0-∞ of 6-MNA after co-administration with pure AN and APE has been observed. Tmax of 6-MNA has been increased to 2 h from 1.5 h in AN + NAB treated group. Changes in mean residential time, clearance and volume of distribution of 6-MNA in APE + NAB treated group and AN + NAB treated group indicated interference of other components of APE other than AN. In pharmacodynamic study, significant decrease in antiarthritic activity of NAB on concomitant administration with APE and AN has been observed. The study concludes that NAB exhibits pharmacokinetic and pharmacodynamic interactions with APE and AN in rats thus alarms the concomitant use of herbal preparations containing APE and AN with NAB. Further study is needed to understand the mechanism and predict the herb-drug interaction in humans. Copyright © 2016 John Wiley & Sons, Ltd.

Gel Formulation of Nabumetone and a Newly Synthesized Analog: Microemulsion as a Photoprotective Topical Delivery System.

Photostability studies were performed on topical formulations containing the anti-inflammatory drug Nabumetone and an analog newly synthesized in order to achieve better photostability and pharmacokinetic profile. Stability tests, according to the International Conference on Harmonization rules, were applied on ethanol solutions and topical gel formulations of both compounds. The photodegradation profiles were monitored by Multivariate curve resolution applied to the UV spectral data. The inclusion of the compounds in microemulsion was investigated to improve light stability and, at the same time, to ensure a sustained release system for skin delivery. All the formulations in solution, gel, microemulsion, and microemulsion-in-gel were exposed to a forced irradiation of 350 W/m2, corresponding to a 21 kJ/m2 min, for up to 300 min. Photostability increased significantly for both drugs in the liquid microemulsion and microemulsion-in-gel, compared to the ethanol solution and plain gel, reaching a residual drug of 97% and 98% for Nabumetone and analog in microemulsion-in-gel, respectively. Permeation experiments on the microemulsion-in-gel showed a better performance of the analog formulated at 0.2%, compared to the same formulation of Nabumetone at 0.7%. These results highlight the potential of the designed matrices as delayed drug delivery systems along with the use of lower drug doses leading to reduced side effects.

Design of a pseudo stir bar sorptive extraction using graphenized pencil lead as the base of the molecularly imprinted polymer for extraction of nabumetone.

Molecularly imprinted polymer (MIP) was synthesized through the coprecipitation method on the graphene oxide anchored pencil lead as a substrate for the first time and applied as an efficient sorbent for pseudo stir bar sorptive extraction of nabumetone. The extracted analyte was determined by a novel spectrophotometric method based on the aggregation of silicate sol-gel stabilized silver nanoparticles in the presence of the analyte. The synthesized polymer was characterized using Fourier transform infrared spectroscopy and field emission scanning electron microscopy. Optimization of important parameters affecting the extraction efficiency was done using central composite design whereas the spectrophotometric method was optimized via one at a time variable. Under the optimal conditions, the calibration curve exhibited linearity in the concentration range of 1.5-20.0 µg L-1. A limit of detection of 0.20 µg L-1, an enhancement factor of 393 and relative standard deviations (at 10 µg L-1, n = 6) of 4.6% and 8.1% for intra- and inter-day analysis were obtained. The developed procedure was successfully utilized for the quantification of traces of nabumetone in tap water and biological samples with the complex matrix including human urine and serum.

Development of Microemulsion Based Nabumetone Transdermal Delivery for Treatment of Arthritis.

BACKGROUND: Nabumetone is biopharmaceutics classification system (BCS) class II drug, widely used in the treatment of osteoarthritis and rheumatoid arthritis. The most frequently reported adverse reactions for the drug involve disturbance in gastrointestinal tract, diarrhea, dyspepsia and abdominal pain. Microemulgel has advantages of microemulsion for improving solubility for hydrophobic drug. Patent literature had shown that the work for drug has been carried on spray chilling, enteric coated tablet, and topical formulation which gave an idea for present research work for the development of transdermal delivery. OBJECTIVE: The objective of the present research work was to optimize transdermal microemulgel delivery for Nabumetone for the treatment of arthritis. METHODS: Oil, surfactant and co-surfactant were selected based on solubility study of the drug. Gelling agents used were Carbopol 934 and HPMC K100M. Optimization was carried out using 32 factorial design. Characterization and evaluation were carried out for microemulsion and microemulsion based gel. RESULTS: Field emission-scanning electron microscopy (FE-SEM) study of the microemulsion revealed globules of 50-200 nm size. Zeta potential -9.50 mV indicated good stability of microemulsion. Globule size measured by dynamic light scattering (zetasizer) was 160nm. Design expert gave optimized batch as F7 which contain 0.2% w/w drug, 4.3% w/w liquid paraffin, 0.71% w/w tween 80, 0.35% w/w propylene glycol, 0.124% w/w Carbopol 934, 0.187% w/w HPMC K100M and 11.68% w/w water. In-vitro diffusion study for F7 batch showed 99.16±2.10 % drug release through egg membrane and 99.15±2.73% drug release in ex-vivo study. CONCLUSION: Nabumetone microemulgel exhibiting good in-vitro and ex-vivo controlled drug release was optimized.

Production of nabumetone nanoparticles: Effect of molecular weight, concentration and nature of cellulose ether stabiliser.

The ability of a range of hydrophilic nonionic cellulose ethers (CEs) (namely methylhydroxethylcellulose, hydroxypropylmethylcellulose, ethylhydroxyethylcellulose, hydroxyethylcellulose and hydroxypropylcellulose) to prepare stable nabumetone nanoparticles (<1000nm, as measured by laser diffraction) using wet-bead milling has been investigated. Due to the limited range of CE molecular weights commercially available, the CEs were degraded using ultrasonication for varying lengths of time to yield CEs of lower molecular weight. Of the CEs tested, only hydroxyethylcellulose was found not to stabilise the production of nabumetone nanoparticles at any of the molecular weights tested, namely viscosity average molecular weights (Mv) in the range of 236-33kg/mol. All other CEs successfully stabilised nabumetone nanoparticles, with the lower molecular weight/viscosity polymers within a series being more likely to result in nanoparticle production than their higher molecular weight counterparts. Unfortunately due to the nature of the ultrasonication process, it was not possible to compare the size of nabumetone particles produced using polymers of identical Mv. There was, however, enough similarity in the Mv of the various polymers to draw the general conclusion that there was no strong correlation between the Mv of the various polymers and their ability to produce nanoparticles. For example hydroxypropylcellulose of 112.2kg/mol or less successfully produced nanoparticles while only ethylhydroxyethylcellulose and hydroxypropylmethyl polymers of 52 and 38.8kg/mol or less produced nanoparticles. These results suggest that polymer molecular weight is not the only determinant of nanoparticle production and that structure of the polymer is at least as important as its molecular weight. In particular the hydrophobic nature of the CE was thought to be an important factor in the production of nabumetone nanoparticles: the more hydrophobic the polymer, the stronger its interaction with nabumetone and the greater its ability to produce nanoparticles. In this context HPC was the most hydrophobic polymer and HEC the least hydrophobic.

Effect of nabumetone on humoral immune responses in mice / Efeito da nabumetona nas respostas imunes humorais em camundongos

Nabumetone is used to reduce the pain and inflammation in rheumatoid arthritis. In the current study, immunomodulatory effect of Nabumetone is investigated in mice. The control group was administered normal saline orally as placebo. Nabumetone was administered orally via gavage in two treatment groups at 14mg/kg.b.w. doses and 28mg/kgb.w., respectively. Haemagglutination (HA) assay, Jerne hemolytic plaque and mice lethality assays were applied. In HA assay, the titer was significantly decreased in Nabumetone treatment groups (P< 0.001). In Jerne hemolytic plaque formation assay, there was a significant reduction (P< 0.001) in number of plaques in Nabumetone treated groups when compared with control. In mice lethality assay, there was a significant difference in mortality ratio of mice in control and Nabumetone treated groups (P< 0.001). Therefore, it is concluded that Nabumetone suppresses the humoral immune response in mice.(AU)

A nabumetona é usada na redução da dor e inflamação da artrite reumática. No presente estudo, o efeito imunomodulador é investigado em camundongos. O grupo de controle recebeu solução salina via oral como placebo. Nabumetona foi administrada oralmente via gavagem em dois grupos de tratamentos com doses de 14mg/kg.b.w. e 28mg/kgb.w., respectivamente. Foram realizados ensaios de hemaglutinação (HA), placa hemolítica de Jerne e letalidade dos camundongos. No ensaio HA, o grau foi significativamente menor nos grupos de tratamento com nabumetoma (P< 0.001). No ensaio de formação de placa hemolítica de Jerne houve redução significativa (P< 0.001) no número de placas em grupos tratados com nabumetoma comparado ao controle. No ensaio de letalidade dos camundongos houve diferença significativa no grau de mortalidade de camundongos no grupo de controle e grupos tratados com nabumetoma (P< 0.001). Portanto, conclui-se que a Nabumetoma suprime a resposta imune humoral em camundongos.(AU)

Vortex-assisted surfactant-enhanced emulsification microextraction based on solidification of floating organic drop combined with high performance liquid chromatography for determination of naproxen and nabumetone.

A novel, rapid, simple and green vortex-assisted surfactant-enhanced emulsification microextraction method based on solidification of floating organic drop was developed for simultaneous separation/preconcentration and determination of ultra trace amounts of naproxen and nabumetone with high performance liquid chromatography-fluorescence detection. Some parameters influencing the extraction efficiency of analytes such as type and volume of extractant, type and concentration of surfactant, sample pH, KCl concentration, sample volume, and vortex time were investigated and optimized. Under optimal conditions, the calibration graph exhibited linearity in the range of 3.0-300.0ngL(-1) for naproxen and 7.0-300.0ngL(-1) for nabumetone with a good coefficient of determination (R(2)>0.999). The limits of detection were 0.9 and 2.1ngL(-1). The relative standard deviations for inter- and intra-day assays were in the range of 5.8-10.1% and 3.8-6.1%, respectively. The method was applied to the determination of naproxen and nabumetone in urine, water, wastewater and milk samples and the accuracy was evaluated through recovery experiments.

Drug-Drug Interactions within Protein Cavities Probed by Triplet-Triplet Energy Transfer.

A new direct and noninvasive methodology based on transient absorption spectroscopy has been developed to probe the feasibility of drug-drug interactions within a common protein binding site. The simultaneous presence of (R)-cinacalcet (CIN) and (S)-propranolol (PPN) within human or bovine α1-acid glycoproteins (AAGs) is revealed by detection of (3)CIN* as the only transient species after laser flash photolysis of CIN/PPN/AAG mixtures at 308 nm. This is the result of triplet-triplet energy transfer from (3)PPN* to CIN, which requires close contact between the two drugs within the same biological compartment. Similar results are obtained with nabumetone and CIN as donor/acceptor partners. This new methodology can, in principle, be extended to a variety of drug/drug/biomolecule combinations.